SUMMARY Energy‐filtering transmission electron microscopy has been applied to the quantification of area fractions of calcium‐containing cytochemical reaction products in central nervous tissue and the retina of fish. The method of electron spectroscopic imaging using electrons with an energy loss of 250 eV produces images with a very high, structure‐sensitive contrast. This is a suitable imaging condition for the reliable detection of reaction products and structural details in unstained ultrathin sections. The images were recorded with a sensitive TV camera and evaluated with the integrated digital image‐analysis system of the Zeiss CEM 902 energy‐filtering electron microscope. An empirical procedure was developed which objectively detects reaction products and calculates characteristic values, taking into account different staining intensities. This new and sensitive method enabled an assessment to be made of the influence of temperature and light adaptation on cytochemically detectable calcium in nervous tissue of fish. Higher amounts of calcium‐containing reaction product were detected in synaptic clefts of the optic tectum in warm‐adapted fish than in cold‐adapted fish. In synaptic vesicles of photoreceptor cells in the fish retina, higher amounts of reaction product were found in dark‐adapted fish than in light‐adapted fish.

SUMMARY The energy‐filtering electron microscopical modes of electron energy‐loss spectroscopy (EELS) and electron spectroscopic imaging (ESI) have been applied to the cytochemical detection of Ca2+ ‐ATPase activity in synaptic terminals in the brain of a cichlid fish. Using a recently developed modification of an enzyme‐histochemical method, cerium phosphate was precipitated as a marker of high‐affinity Ca2+ ‐ATPase activity. This is considered to be a marker for the plasmalemma‐bound calcium pump, an enzyme which plays a crucial role in the regulation of the cytoplasmic calcium concentrations and therefore of the reactivity of nerve cells. High‐affinity Ca2+ ‐ATPase activity is located preferentially at the inner side of synaptic plasma membranes and enables a discrimination of different types of synapse. It is only by using EELS and ESI that the very small amounts of high‐affinity Ca2+ ‐ATPase reaction product can be analysed reliably and located precisely. These new electron microscopical techniques offer powerful tools for cytochemical studies.

Cytochrome oxidase activity was evaluated cytochemically in rod photoreceptor cells in the retina of the cichlid fish Oreochromis mossambicus after different stimulation protocols. The enzyme activity was assessed semiquantitatively by estimating the volume ratio of mitochondria classified according to the intensity of enzyme reactivity. Dark adaptation for 5 hr induced an increase of cytochrome oxidase activity both in vivo and in vitro, i.e., in isolated retinas. Short-term illumination (1 hr) of isolated retinas adapted previously in vivo to darkness caused a significant decrease of enzyme activity, whereas short-term darkening after in vivo light adaptation had no effect. Chemical stimulation for 15 min with increased K+ concentration (20 mM) reduced the enzyme activity, i.e., chemical depolarization did not have the same effect as depolarization induced by darkening. Significant changes in cytochrome oxidase activity were apparent within 1 hr of stimulation, so that this method for analysis of neuronal activity can be applied even in short-term experiments.